WO2021216757A1 - Inhibiteurs de la multimérisation de protéine de liaison à l'arn et leurs procédés d'utilisation - Google Patents

Inhibiteurs de la multimérisation de protéine de liaison à l'arn et leurs procédés d'utilisation Download PDF

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WO2021216757A1
WO2021216757A1 PCT/US2021/028460 US2021028460W WO2021216757A1 WO 2021216757 A1 WO2021216757 A1 WO 2021216757A1 US 2021028460 W US2021028460 W US 2021028460W WO 2021216757 A1 WO2021216757 A1 WO 2021216757A1
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compound
sri
substituted
unsubstituted
hur
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PCT/US2021/028460
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Peter King
Louis B. NABORS
Natalia FILIPPOVA
Xiuhua YANG
Subramaniam Ananthan
Vibha Pathak
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The Uab Research Foundation
Southern Research Institute
United States Government As Represented By The Department Of Veterans Affairs
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Priority to CA3176337A priority Critical patent/CA3176337A1/fr
Priority to US17/996,886 priority patent/US20230159542A1/en
Publication of WO2021216757A1 publication Critical patent/WO2021216757A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • RNA-binding protein Human antigen R protein serves as such a node.
  • HuR functionality in cancer cells is strictly dependent on HuR nuclear/cytoplasmic shuttling and dimerization.
  • the pathological processes driven by HuR promote cancer and inflammation.
  • HuR belongs to the mRNA-binding proteins of the ELAV family and is a chemotherapeutic target for many types of cancer.
  • HuR RNA-binding protein Human antigen R protein
  • the compounds have a high affinity for HuR multimers and inhibit pathological processes at micro- to nano- molar ranges.
  • the compounds provide a highly unique and powerful therapeutic option for numerous disease processes related to neoplastic progression or acute/chronic inflammation.
  • compounds of the following formula are provided: or a pharmaceutically acceptable salt or prodrug thereof, wherein is a single bond or a double bond;
  • R 1 , R 2 , and R 3 are each independently selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted
  • R 5 is optionally substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted alkyl.
  • R 6 is hydrogen or C 1 -C 6 alkyl and/or R 7 is hydrogen, C 1 -C 6 alkyl, or acetyl.
  • the compound can optionally have the following formula:
  • the compound is selected from the group consisting of:
  • R 4 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted alkyl.
  • R 6 is hydrogen or C 1 -C 6 alkyl and/or R 7 is hydrogen, C 1 -C 6 alkyl, or acetyl.
  • R 1 , R 2 and R 3 are H.
  • the compound is optionally selected from the group consisting of:
  • pharmaceutical compositions comprising a compound as described herein and a pharmaceutically acceptable carrier.
  • kits comprising a compound or a pharmaceutical composition as described herein are provided. Further described herein are methods of treating or preventing cancer in a subject, comprising administering to a subject an effective amount of a compound or a composition as described herein.
  • the cancer is brain cancer or glioma.
  • the methods can further comprise administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent).
  • a second therapeutic agent e.g., a chemotherapeutic agent.
  • methods of inhibiting HuR multimerization in a cell comprising contacting a cell with an effective amount of a compound or a composition as described herein. The contacting can be performed in vitro or in vivo.
  • methods of treating or preventing inflammation in a subject comprising administering to a subject an effective amount of a compound or a composition as described herein.
  • the inflammation is neuroinflammation.
  • the compound is optionally selected from the group consisting of: Also described herein are method of inhibiting HuR multimerization in a cell, comprising contacting a cell with an effective amount of a compound or composition as described herein. The contacting can be performed in vivtro or in vivo. Further described herein are methods of treating or preventing pain in a subject suffering from spinal cord injury, comprising administering to a subject an effective amount of a compound or a composition as described herein. Optionally, the pain is neuropathic pain.
  • the compound is optionally selected from the group consisting of: Also described herein are method of inhibiting HuR multimerization in a cell, comprising contacting a cell with an effective amount of a compound or composition as described herein.
  • FIG. 1A is an illustration of Kaplan-Meier overall survival curves demonstrating statistically significant differences between “high ELAVL1 expression” (indicated as “high”) versus “low ELAVL1 expression” (indicated as “low”) for grouping of all brain tumors.
  • Figure 1B is an illustration of ELAVL1 (left graph) and ACTB (as a control; right graph) mRNA expression in the brain tumor samples versus normal samples.
  • Figure 1C is an illustration of Kaplan-Meier overall survival curves according to the tracks “high ELAVL1 expression” (indicated as “high”) versus “low ELAVL1 expression” (indicated as “low) for tumor subsets of grades 1-2 (left graph), grade 3 (middle graph) and grade 4 (right graph).
  • Figure 1D is an illustration of the enhancemement of ELAVL1/ACTB mRNA ratios with the increase of the brain tumor grade with results shown as mean ⁇ SD.
  • Figure 1E is an illustration of the gene tests exhibiting significant positive or negative correlations with the ELAVL1 exzpression for normal brain and grades 1-4 tumors.
  • Figure 2A is an illustration of a cell-based assay used herein.
  • Figure 2B is an illustration of the control portion of a cell-based assay used herein.
  • Panel A shows the structure of compound A92 and a dose-response graph showing the inhibition of HuR dimerization for compound SRI-41964 (A-92).
  • Panel B shows the structure of SRI-41664 and a dose-response graph showing the inhibition of HuR dimerization for compound SRI-41664.
  • Panel C shows the structures of SRI-41964, SRI-42124, SRI-41664, and SRI-42127 and a graph of dose-response graph showing the inhibition of HuR dimerization for compound SRI-42127.
  • Figure 4 is a graph of dose-response curves showing the inhibition of HuR dimerization for SRI-42127 in four independent cell clones.
  • Figure 5A contains graphs illustrating cell-viability dose responses for compounds of this disclosure.
  • Figure 5B contains low cytometry plots illustrating cell cycle distribution in cell lines as well as a bar graph representing the average number of cells (mean ⁇ SD, %) in each phase of the cell cycle after treatment with SRI-424127 compound normalized to the corresponding control values (vehicle treatment).
  • Figure 5C contains phase-contrast images illustrating primary human neurons after treatment with SRI-42127 (48 hours) versus control (vehicle treatment). Scale bar, 100 um.
  • Figure 5D is a graph illustrating that neuron numbers were not affected by treatment with SRI-42127.
  • Figure 5E is a graph illustrating the metabolic alterations in primary human neurons after treatment with SRI-42127.
  • Figure 5F is a graph illustrating the metabolic alterations in primary human neurons and astrocytes after treatment with SRI-42127 for 48 hours along with representative images of cleaved caspase-3 immunostaining after SRI-42127 treatment versus control (vehicle) for neurons where DAPI marks the cell nucleus.
  • Figure 6 Panel A is an illustration of U251 glioma cells with dox-inducible HuR- dimerization reporters and dox-indicuble overexpression of the control Fluc reporter.
  • Panel A shows a decrease in HuR-dimerization as a function of higher concentrations of SRI-42127 over 6 hours (left graph) and a decrease in cell viability as a function of higher concentrations of SRI-42127 over 48 hours (right graph).
  • Figure 6 Panel B shows no change with increasing concentrations of SRI-42127 over 6 hours (left graph) and a decrease in cell viability as a function of higher concentrations of SRI-42127 over 48 hours (right graph).
  • Figure 7 Panel A depicts representative western blots illustrating HuR expression in the cytoplasmic fraction of PDGx cell lines.
  • FIG 7 Panel B depicts a representative western blot illustrating HuR multimerization in the cytoplasmic fraction of PDGx cell lines detected in non-reducing and non-denaturing conditions versus reducing and denaturing conditions.
  • Figure 7 depicts a representative western blot illustrating HuR expression in the established cell lines compared to the HuR expression in the PDGx XD456 cell line.
  • Figure 7 depicts representative western blots illustrating HuR nuclear/cytoplasmic distribution in the PDGx and established glioma cell lines using anti- Lamin A/C antibody to confirm nuclear fraction, and anti- ⁇ Tub antibody to confirm cytoplasmic fraction.
  • Panel E shows a graph illustrating IC 50 S of SRI-424127 in several cell lines.
  • Figure 8A is a bar graph illustrating the decrease of Bcl2/18S and Mcl1/18S mRNAs after treatment with compounds of this disclosure.
  • Figure 8B is a western blot confirming reduction of Bcl2 and Mcl1 proteins in cytoplasmic fractions of U251 and XD456 cell lines after treatment.
  • Figure 8C is a western blot illustrating appearances of cleaved PARP and cleaved caspase 3 in glioma cell lines after treatment with compounds of this disclosure.
  • Figure 8D contains bar graphs illustrating the significant reduction of colony formations in U251 and XD456 cell lines after treatment with compounds of this disclosure, and photographs of soft agar colony formation assays.
  • Figure 9A is a photograph of luminescent imaging of glioma progression in mice with and without SRI-42127 treatment with bar graphs illustrating luminescence signals detected from intracranial tumors on the second (top) and third (bottom) weeks of mouse treatment.
  • Figure 9B illustrates immunostaining for HuR on tumor brain tissue for mouse groups with and without SRI-42127 treatment.
  • Figure 9C illustrates immunostaining for Bcl2 on a tumor brain tissue for mouse groups with and without SRI-42127 treatment.
  • Figure 9D illustrates immunostaining for MCl1 on a tumor brain tissue for mouse groups with and without SRI-42127 treatment.
  • Figure 9E illustrates the computational docking of SRI-42127 at HuR.
  • Panel A contain cluster charts of enrichment of down-regulated genes and targeted sub-cellular structures.
  • Panel B contain cluster charts of enrichment of up-regulated genes and targeted sub-cellular structures.
  • Panel A contains a bar graph and images of HuR localization within microglial cells in unstimulated state (DMSO), stimulated state (LPS+), and in stimulated state (LPS+) with SRI-42127.
  • Panel B is a graph illustrating microglia viability in the presence of different concentration of SRI-42127.
  • Panel C is a set of images of microglia cells immunostained to assess the specificity of SRI-42127 for blocking HuR cytoplasmic translocation by assessing SRI-42127 effects on HMGB1 translocation.
  • FIG. 11 Panel D contains bar graphs illustrating the nuclear/cytoplastmic (N/C) ratio for HuR and HMGB1.
  • Figure 12 contains bar graphs illustrating levels of inflammatory cytokines, chemokines, and other mediators quantified by qPCR of mRNAs harvested from cultured primary microglial cells isolated from neonatal mouse brains that were activated (LPS+) and then treated with varying doses of SRI-42127 or vehicle for a 24 hour period.
  • Figure 13 contains bar graphs illustrating attenuation of secreted protein product by varying concentrations of SRI-42127.
  • the left bar (or left-most position) in each grouping represents the absence of LPS and the absence of SRI-42147 or any vehicle.
  • the second bar (or second position) in each grouping represents the presence of LPS and a vehicle.
  • the third bar (or third position) in each grouping represents the presence of LPS and 0.05 ⁇ M SRI- 42127.
  • the fourth bar (or fourth position) in each grouping represents the presence of LPS and 0.1 ⁇ M SRI-42127.
  • the fifth bar (or fifth position) in each grouping represents the presence of LPS and 0.5 ⁇ M SRI-42127.
  • the sixth bar (or sixth position) in each grouping represents the presence of LPS and 1.0 ⁇ M SRI-42127.
  • Figure 14 is a western blot of microglial lysates showing attenuation of protein presence with modest but significant attenuation of HuR and another RNA binding protein, KSRP.
  • the set of graphs are a quantitative depiction of the western blot data.
  • Panel A is a set of images of cultured astrocytes that were activated (LPS+) in the presence of vehicle or SRI-42127 that were then fixed and immunostained with GFAP and HuR antibodies to analyze HuR localization.
  • the bar graph on the right of Panel A quantitates the HuR N/C ratio for astrocytes in the presence of DMSO, activated with LPS in vehicle, and activated with LPS with SRI-42127.
  • Panel B is a bar graph illustrating the impact of SRI-42127 on induction of inflammatory cytokine mRNAs.
  • Figure 16 contains bar graphs illustrating suppression of inflammatory cytokine, chemokine, and other mediators mRNA at varying doses of SRI-42127.
  • Panel A contains two bar graphs illustrating attenuation of monocyte (top) and neutrophil (bottom) migration of cells harvested from murine bone marrow and placed in an upper chamber to conditioned media from LPS-activated microglia placed in a lower chamber, treated with varying doses of SRI-42127 where migration across the transwell was quantitated by staining the filter with hematoxylin.
  • Panel B contains two bar graphs illustrating attenuation of monocyte (top) and neutrophil (bottom) migration of cells harvested from murine bone marrow and placed in an upper chamber to conditioned media from LPS-activated astroglia placed in a lower chamber, treated with varying doses of SRI-42127 where migration across the transwell was quantitated by staining the filter with hematoxylin.
  • Panel C contains a bar graph and images of suppression of migration of untreated microglia toward conditioned media from SRI-42127-treated microglia.
  • Figure 18 contains photomicrographs following translocation of HuR in the microglial nucleus or cytoplasm and a bar graph showing the quantitation by way of N/C ratio for untreated and treated mice.
  • Figure 19 Panel A is a set of images illustrating the activation state of microglia by measuring the fluorescent intensity of IBA1 stained microglia in the presence of vehicle and SRI-42127.
  • Figure 19 Panel B is a bar graph illustrating the attenuating effects of SRI-42127 in IBA1 intensity.
  • Panel 19 is a bar graph illustrating the number of cells per HPF evaluated between control and treated mice brain samples.
  • Panel A is a cartoon illustrating the three brain sections of interest, frontal brain, middle brain, and hindbrain.
  • Figure 20, Panel B is a plot illustrating flow cytometry results from neutrophils and monocytes using Ly6c and Ly6g markers on CD11+ sorted cells.
  • Panel C is a set of bar graphs illustrated as %CD11b+ cells showing significant attenuation of infiltrating neutrophils (left) and monocytes (right) in whole brain, front brain, middle brain, and hindbrain of treated and untreated mice.
  • Panel D is a set of bar graphs for HuR KO mice illustrated as %CD11b+ cells showing significant attenuation of infiltrating monocytes (right), but not neutrophils (left), in whole brain, front brain, middle brain, and hindbrain of treated and untreated mice.
  • Panel C represents a plot showing Von Frey testing at 1 week depiciting a large increase in withdrawal threshold indicating reduced allodynia for mice that underwent spared nerve injury (sciatic nerve) and were then treated with SRI-42127 (10 mg/kg i.p.) every 6 hours for 4 days.
  • Panel D is a plot showing that SRI-42127 significantly reversed allodynia at 30 and 60 min post injection. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001) for vehicle (DMSO) treated group which received either an injection of SRI-42127 (10 mg/kg) or vehicle at 1 week.
  • the compounds disclosed herein are small molecule inhibitors of the post- transcriptional regulator HuR, which is a critical control point for genes related to proliferation, angiogenesis, invasion, cell death, immune evasion and inflammation, genomic instability, and cell immortality. HuR moves into the cytoplasm and form multimers, which ensures a disease promoting genotype.
  • the small molecules described herein inhibit the multimerization step, thus reversing a pathological state in a subject.
  • Compounds A class of compounds described herein includes Formula I: and pharmaceutically acceptable salts or prodrugs thereof. In Formula I, is a single bond or a double bond.
  • R 1 , R 2 , and R 3 are each independently selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, alkoxy, aryloxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • R 4 and R 5 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • R 4 is hydrogen.
  • R 5 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted alkyl.
  • Y 1 , Y 2 , and Y 3 are each independently selected from NR 6 and CR 7 .
  • R 6 and R 7 are each independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbonyl.
  • at least two of Y 1 , Y 2 , and Y 3 are NR 6 .
  • R 6 is hydrogen or C 1 -C 6 alkyl and/or R 7 is hydrogen, C 1 -C 6 alkyl, or acetyl.
  • the compound is not A-92 (also called SRI-41964), which has the following structure:
  • the compounds according to Formula I are represented by Structure I-A: Structure I-A In Structure I-A, , R 1 , R 2 , R 3 , R 4 , R 5 , Y 1 , Y 2 , and Y 3 are as defined above for Formula I.
  • the compounds according to Structure I-A are selected from the following: Structure I-A1 Structure I-A2 Structure I-A3 In Structure I-A1, Structure I-A2, and Structure I-A3, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are as defined above for Formula I.
  • R 4 is hydrogen.
  • R 5 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted alkyl.
  • R 6 is optionally hydrogen or C 1 -C 6 alkyl and/or R 7 is optionally hydrogen, C 1 -C 6 alkyl, or acetyl.
  • Structure I-A examples include the following compounds: In some cases, the compounds according to Formula I are represented by Structure I-B: Structure I-B In Structure I-B, , R 1 , R 2 , R 3 , R 4 , Y 1 , Y 2 , and Y 3 are as defined above for Formula I.
  • the compounds according to Structure I-B are selected from the following: Structure I-B1 Structure I-B2 Structure I-B3 In Structure I-B1, Structure I-B2, and Structure I-B3, R 1 , R 2 , R 3 , R 4 , R 6 , and R 7 are as defined above for Formula I.
  • R 4 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted alkyl.
  • R 6 is hydrogen or C 1 -C 6 alkyl and/or R 7 is hydrogen, C 1 -C 6 alkyl, or acetyl.
  • R 1 , R 2 and R 3 are H.
  • each of R 1 , R 2 and R 3 is H.
  • Examples of Structure I-B include the following compounds: Specific examples of compounds of this disclosure include: Compound 2 (SRI-42126) Compound 3 (SRI-42124) Compound 4 (SRI-42125) Compound 13 (SRI-42916) Compound 14 (SRI-42917) Compound16 (SRI-43935) Compound 17 (SRI-43936) Compound 18 (SRI-43937) Compound 19 (SRI-42127) Compound 20 (SRI-43372) Compound 21 (SRI-43369) Compound 22 (SRI-42719) Compound 23 (SRI-43413) Compound 24 (SRI-43175) Compound 25 (SRI-42918) Compound 26 (SRI-43566) Compound 27 (SRI-43499) Compound 28 (SRI-43568) Compound 29 (SRI-43753) Compound 30 (SRI-43411) Compound 31 (SRI-43371) Compound 32 (SRI-43264) Compound 33 (SRI-43263) Compound 34 (SRI
  • Compound 53 (SRI-44048) Compound 54 (SRI-44049).
  • Compound 55 (SRI-44000) Compound 56 (SRI-44001) Compound 57 (SRI-44002) and Compound 58 (SRI-43266).
  • the compound is not: Compound A-92 (SRI-41964) Compound 26 (SRI-43566) Compound 27 (SRI-43499) Compound 31 (SRI-43371) Compound 32 (SRI-43264) Compound 41 (SRI-43497) or Compound 42 (SRI-43494).
  • the compounds are selected from the following: Compound 59 (SRI-41639) Compound 60 (SRI-41646) Compound 61 (SRI-41647) Compound 62 (SRI-42123) Compound 63 (SRI-42219) Compound 64 (SRI-43183) Compound 65 (SRI-43414) Compound 66 (SRI-43415) Compound 67 (SRI-43646) Compound 68 (SRI-43649) Compound 69 (SRI-43650) Compound 70 (SRI-43651) Compound 71 (SRI-43652) Compound 72 (SRI-43653) Compound 73 (SRI-43654) and Compound 74 (SRI-43655).
  • alkyl, alkenyl, and alkynyl include straight- and branched- chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups useful with the compounds and methods described herein include C 1 -C 20 alkyl, C 2 -C 20 alkenyl, and C 2 -C 20 alkynyl.
  • Additional ranges of these groups useful with the compounds and methods described herein include C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, and C 2 -C 4 alkynyl.
  • Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly as alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the backbone.
  • Ranges of these groups useful with the compounds and methods described herein include C 1 -C 20 heteroalkyl, C 2 -C 20 heteroalkenyl, and C 2 -C 20 heteroalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C 1 - C 12 heteroalkyl, C 2 -C 12 heteroalkenyl, C 2 -C 12 heteroalkynyl, C 1 -C 6 heteroalkyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, C 1 -C 4 heteroalkyl, C 2 -C 4 heteroalkenyl, and C 2 -C 4 heteroalkynyl.
  • cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. Ranges of these groups useful with the compounds and methods described herein include C 3 -C 20 cycloalkyl, C 3 -C 20 cycloalkenyl, and C 3 -C 20 cycloalkynyl.
  • Additional ranges of these groups useful with the compounds and methods described herein include C 5 -C 12 cycloalkyl, C 5 -C 12 cycloalkenyl, C 5 -C 12 cycloalkynyl, C 5 -C 6 cycloalkyl, C 5 -C 6 cycloalkenyl, and C 5 -C 6 cycloalkynyl.
  • the terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly as cycloalkyl, cycloalkenyl, and cycloalkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the cyclic backbone.
  • Ranges of these groups useful with the compounds and methods described herein include C 3 -C 20 heterocycloalkyl, C 3 -C 20 heterocycloalkenyl, and C 3 -C 20 heterocycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C 5 -C 12 heterocycloalkyl, C 5 -C 12 heterocycloalkenyl, C 5 -C 12 heterocycloalkynyl, C 5 -C 6 heterocycloalkyl, C 5 -C 6 heterocycloalkenyl, and C 5 -C 6 heterocycloalkynyl.
  • Aryl molecules include, for example, cyclic hydrocarbons that incorporate one or more planar sets of, typically, six carbon atoms that are connected by delocalized electrons numbering the same as if they consisted of alternating single and double covalent bonds.
  • An example of an aryl molecule is benzene.
  • Heteroaryl molecules include substitutions along their main cyclic chain of atoms such as O, N, or S. When heteroatoms are introduced, a set of five atoms, e.g., four carbon and a heteroatom, can create an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imadazole, oxazole, pyridine, and pyrazine.
  • Aryl and heteroaryl molecules can also include additional fused rings, for example, benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline.
  • the aryl and heteroaryl molecules can be attached at any position on the ring, unless otherwise noted.
  • alkoxy as used herein is an alkyl group bound through a single, terminal ether linkage.
  • aryloxy as used herein is an aryl group bound through a single, terminal ether linkage.
  • alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkyloxy, and heterocycloalkyloxy are an alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, heteroaryloxy, cycloalkyloxy, and heterocycloalkyloxy group, respectively, bound through a single, terminal ether linkage.
  • hydroxy as used herein is represented by the formula —OH.
  • amine or amino as used herein are represented by the formula —NZ 1 Z 2 , where Z 1 and Z 2 can each be substitution group as described herein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl molecules used herein can be substituted or unsubstituted.
  • the term substituted includes the addition of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group to a position attached to the main chain of the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl, e.g., the replacement of a hydrogen by one of these molecules.
  • substitution groups include, but are not limited to, hydroxy, halogen (e.g., F, Br, Cl, or I), and carboxyl groups.
  • halogen e.g., F, Br, Cl, or I
  • carboxyl groups examples include, but are not limited to, hydroxy, halogen (e.g., F, Br, Cl, or I), and carboxyl groups.
  • the term unsubstituted indicates the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear decane (–(CH 2 ) 9 –CH 3 ).
  • the compounds described herein can be prepared in a variety of ways.
  • the compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry.
  • the compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art. Variations on Formula I and the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups.
  • protecting groups can be found, for example, in Wuts, Greene’s Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.
  • Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of ordinary skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent.
  • Product or intermediate formation can be monitored according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible), mass spectrometry (MS), or high-resolution mass spectrometry (HRMS), or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 C-NMR), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible), mass spectrometry (MS), or high-resolution mass spectrometry (HRMS), or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).
  • HPLC high performance liquid chromatography
  • the compounds described herein or derivatives thereof can be provided in a pharmaceutical composition.
  • the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents.
  • compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier.
  • carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition.
  • physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, NJ).
  • buffers such as phosphate buffers, citrate buffer, and buffer
  • compositions containing the compound described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Isotonic agents for example, sugars, sodium chloride, and the like may also be included.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules.
  • the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example,
  • the dosage forms may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
  • inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl
  • the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
  • Suspensions in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
  • Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, inhalants, and skin patches.
  • the compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be
  • compositions can include one or more of the compounds described herein or pharmaceutically acceptable salts thereof.
  • pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein.
  • salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like
  • non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder.
  • the effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.0001 to about 200 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day.
  • the dosage amount can be from about 0.01 to about 150 mg/kg of body weight of active compound per day, about 0.1 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active compound per day, about 0.01 to about 50 mg/kg of body weight of active compound per day, about 0.05 to about 25 mg/kg of body weight of active compound per day, about 0.1 to about 25 mg/kg of body weight of active compound per day, about 0.5 to about 25 mg/kg of body weight of active compound per day, about 1 to about 20 mg/kg of body weight of active compound per day, about 1 to about 10 mg/kg of body weight of active compound per day, about 20 mg/kg of body weight of active compound per day, about 10 mg/kg of body weight of active compound per day, about 5 mg/kg of body weight of active compound per day, about 2.5 mg/kg of body weight of active compound per day, about 1.0 mg/kg of body weight of active compound per day,
  • the dosage amounts are from about 0.01 mg/kg to about 10 mg/kg of body weight of active compound per day.
  • the dosage amount is from about 0.01 mg/kg to about 5 mg/kg.
  • the dosage amount is from about 0.01 mg/kg to about 2.5 mg/kg.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Further, depending on the route of administration, one of skill in the art would know how to determine doses that result in a plasma concentration for a desired level of response in the cells, tissues and/or organs of a subject. IV. Methods of Use Provided herein are methods of treating or preventing cancer in a subject. The methods include administering to a subject an effective amount of any compound disclosed herein or an effective amount of any composition disclosed herein.
  • the expression “effective amount,” when used to describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example, an amount that results in tumor growth rate reduction. Additional steps can be included in the method described herein.
  • the methods can further include the steps of selecting a subject with cancer, and administering to the subject one or more of the compounds as described herein.
  • the cancer is bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, glioma, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, or testicular cancer.
  • the methods comprise administering to the subject a second therapeutic agent.
  • Additional therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-depressants, anxiolytics, antibodies, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines, chemokines, and/or growth factors.
  • Anti-inflammatory agents that may be administered with the provided compounds or compositions include, but are not limited to, glucocorticoids and the nonsteroidal anti- inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicyclic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S- adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, ninesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and teni
  • a chemotherapeutic agent is a compound or composition effective in inhibiting or arresting the growth of an abnormally growing cell.
  • an agent may be used therapeutically to treat cancer as well as other diseases marked by abnormal cell growth.
  • chemotherapeutic compounds include, but are not limited to, bexarotene, gefitinib, erlotinib, gemcitabine, paclitaxel, docetaxel, topotecan, irinotecan, temozolomide, carmustine, vinorelbine, capecitabine, leucovorin, oxaliplatin, bevacizumab, cetuximab, panitumumab, bortezomib, oblimersen, hexamethylmelamine, ifosfamide, CPT- 11, deflunomide, cycloheximide, dicarbazine, asparaginase, mitotant, vinblastine sulfate, carboplatin, colchicine,
  • anthracyclines such as doxorubicin, liposomal doxorubicin, and diethylstilbestrol doxorubicin, bleomycin, daunorubicin, and dactinomycin
  • antiestrogens e.g., tamoxifen
  • antimetabolites e.g., fluorouracil (FU), 5-FU, methotrexate, floxuridine, interferon alpha-2B, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine
  • cytotoxic agents e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, vincristine and vincristine sulfate
  • hormones e.g., medroxyprogesterone, estram
  • any of the aforementioned therapeutic agents can be used in any combination with the compositions described herein.
  • Combinations are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second)
  • the term combination is used to refer to concomitant, simultaneous, or sequential administration of two or more agents.
  • a compound or therapeutic agent as described herein may be administered in combination with a radiation therapy, an immunotherapy, a gene therapy, or a surgery. Also described herein are method of treating inflammation in a subject.
  • the methods include administering to a subject an effective amount of any compound disclosed herein or an effective amount of any composition disclosed herein. Additional steps can be included in the method described herein. For example, the methods can further include the steps of selecting a subject with inflammation, and administering to the subject one or more of the compounds as described herein.
  • the inflammation can be the result of diseases related to inflammation including but not limited to heart attack, cystic fibrosis (CF), chronic bronchitis, emphysema, bronchiolitis obiterans syndrome (BOS), interstitial pneumonia, Alzheimer’s Disease, congestive heart failure, stroke, arthritis, aortic valve stenosis, rheumatoid arthritis, kidney failure, lupus, asthma, psoriasis, pancreatitis, allergies, fibrosis, surgical complications, anemia, fibromyalgia, chronic obstructive pulmonary disease (COPD), bacterial infection and viral infection and other inflammatory diseases including, but not limited to neuroinflammation.
  • diseases related to inflammation including but not limited to heart attack, cystic fibrosis (CF), chronic bronchitis, emphysema, bronchiolitis obiterans syndrome (BOS), interstitial pneumonia, Alzheimer’s Disease, congestive heart failure, stroke, arthritis, a
  • the methods include administering to a subject an effective amount of any compound disclosed herein or an effective amount of any composition disclosed herein. Additional steps can be included in the method described herein. For example, the methods can further include the steps of selecting a subject with pain or at risk of developing pain (e.g., due to an injury that has occurred, etc.), and administering to the subject one or more of the compounds as described herein.
  • the pain can be neuropathic pain.
  • the pain can be the result of a spinal cord injury.
  • the pain can be the result of chronic nerve injury.
  • the methods and compounds described herein are also useful in inhibiting HuR multimerization in a cell.
  • the methods of inhibiting HuR multimerization in a cell include contacting a cell with a compound as described herein.
  • the method is performed in vitro.
  • the method is performed in vivo.
  • treatment, treat, or treating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or one or more symptoms of the disease or condition.
  • a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs (e.g., size of the tumor or rate of tumor growth) of the disease in a subject as compared to a control.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • the terms prevent, preventing, and prevention of a disease or disorder refers to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder.
  • references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.
  • subject means both mammals and non mammals.
  • kits for treating or preventing cancer in a subject can include any of the compounds or compositions described herein.
  • a kit can include one or more compounds of Formula I.
  • a kit can further include one or more additional agents, such as one or more chemotherapeutic agents.
  • a kit can include an oral formulation of any of the compounds or compositions described herein.
  • a kit can include an intravenous formulation of any of the compounds or compositions described herein.
  • a kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions (e.g., a syringe), and/or a carrier.
  • directions for use of the kit e.g., instructions for treating a subject
  • a container e.g., a means for administering the compounds or compositions
  • a carrier e.g., a syringe
  • the inhibitors of HuR multimer formation as described herein are soluble, have micromolar activity, and penetrate the blood-brain-barrier (BBB). Molecules developed were evaluated in a robust cell-based assay of HuR dimerization for activity validation and specificity.
  • the inhibitor SRI 42127 demonstrates robust activity in vitro across numerous primary patient-derived glioblastoma (PDGx) xenolines with arrest of proliferation, induction of apoptosis, and inhibition of colony formation with a specific inhibition of HuR multimer formation. It has favorable attributes with central nervous system penetration and demonstrates a growth inhibitor benefit in mouse glioma models.
  • RNA and protein analysis following exposure of a panel of PDGx xenolines across glioblastoma molecular subtypes confirms inhibition of HuR targets and their corresponding signaling pathways.
  • This work provides direct evidence of the anti-cancer effects of HuR inhibition and generates a molecule for further development exploiting a novel oncogenesis dependent interaction.
  • the new class of inhibitor compounds has proven to be a powerful RNA- binding protein multimerization inhibitor in the treatment of inflammatory diseases.
  • the inhibitor SRI-42127 demonstrates a significant dose-dependent decrease in cells with high cytoplasmic HuR without toxicity to the cells within the tested range.
  • SRI-42127 blocks HuR cytoplasmic translocation in activated microglia and suppresses production of pro-inflammatory mediators in activated microglia. Further, SRI-42127 suppresses production of pro-inflammatory mediators in activated astroglia and suppresses chemoattraction of monocytes and neutrophils to microglial and astrogial secreted signals. Finally, systemic administration of SRI-42127 also suppresses HuR translocation in microglia in vivo and neuroinflammatory responses to lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • this work also provides direct evidence of the anti-inflammatory effects of HuR inhibition as it relates to neuroinflammation and presents SIR-42127 as a molecule for further development in the treatment of neuroinflammation in particular, as well as secondary tissue injury related to trauma.
  • this work also provides direct evidence that SRI-42127 exerts a neuroprotective effect in spinal cord injury and an anti-neuropathic pain effect in chronic nerve injury.
  • Example 1 Synthesis of Compounds All reactions were carried out in an oven- or flame-dried glassware under argon atmosphere using gas-tight syringe, cannula, and septa. The reaction temperatures were measured externally.
  • Proton NMR spectra were recorded on a Varian 400-MR NMR spectrometer operating at 400 MHz calibrated to the solvent peak and TMS peak.
  • High-resolution mass spectra were recorded using an Agilent 6210 time-of- flight mass spectrometer.
  • the purity of the final compounds was checked by analytical HPLC using an Agilent 1100 LC system equipped with Phenomenex Kinetex C18 (5 ⁇ m, 4.6 x 150 mm) column and a diode array detector (DAD) monitoring at multiple wavelengths, using the solvent system A: H2O, B: CH 3 CN, linear gradient from 5-95% B over 20 min at a flow rate 1.0 mL/min.
  • the mixture was degassed with argon for 10 minutes and then PdCl 2 (dppf).CH 2 Cl 2 adduct (173 mg, 0.21 mmol) was added.
  • the reaction mixture was heated at 90 °C (oil bath temp) in a sealed screw capped reactor for 18 hours.
  • the reaction mixture was diluted with EtOAc (100 mL) and then stirred at 20 °C for 30 minutes.
  • the mixture was filtered through Celite and the Celite pad was rinsed with EtOAc (20 mL).
  • the combined filtrate was washed with saturated sodium bicarbonate (NaHCO3; 2 x 50 mL), saturated NH 4 Cl (50 mL), followed by brine (100 mL).
  • This intermediate was prepared using the following general procedure (Method C) for displacement of chloro with amine.3-Bromo-6-chloroimidazo[1,2- b]pyridazine (2 g, 8.6 mmol), and N-methyltetrahydro-2H-pyran-4-amine (1.9 g, 17.2 mmol) and cesium carbonate (3.4 g, 10.3 mmol) in DMF (4 mL) were placed in a Biotage 20 mL- microwave vial equipped with a stirrer. The mixture was heated in the microwave reactor for 4 hours at 180 °C.
  • Step 2 The above intermediate was reacted with (1H-indazol-5-yl)boronic acid using Method B to obtain the desired product. Yield 22%.
  • TLC R f 0.40 (CHCl 3 -MeOH, 9:1).
  • Step 2 Reaction of the above intermediate with (1H-indazol-6-yl)boronic acid using Method B afforded the desired target compound 22. Yield 31%.
  • TLC R f 0.40 (CHCl 3 - MeOH, 9:1).
  • Step 2 The above intermediate was reacted with (1H-indazol-6-yl)boronic acid using Method B to obtain the target compound 25. Yield 13%.
  • TLC R f 0.40 (CHCl 3 -MeOH, 9:1).
  • This intermediate was prepared from 3-bromo-6-chloroimidazo[1,2-b]pyridazine and tetrahydro-2H-pyran-4-amine using Method D.
  • Step 2 3-Bromo-N-ethyl-N-(tetrahydro-2H-pyran-4-yl)imidazo[1,2-b]pyridazin- 6-amine.
  • This intermediate was prepared by the following general procedure (Method F) for N-alkylation.
  • This intermediate was prepared from 3-bromo-6-chloroimidazo[1,2-b]pyridazine and tetrahydro-2H-pyran-4-amine using Method D.
  • Step 2 3-Bromo-N-propyl-N-(tetrahydro-2H-pyran-4-yl)imidazo[1,2- b]pyridazin-6-amine.
  • This intermediate was prepared by reacting the product obtained in Step 1 with 1-iodopropane using Method F. Yield 88%.
  • TLC Rf 0.30 (CHCl3-MeOH, 9:1).
  • Step 3 The above intermediate was reacted with (1H-indazol-6-yl)boronic acid using Method B. Yield 38%.
  • TLC R f 0.35 (CHCl 3 -MeOH, 9:1).
  • Step 1 3-Bromo-N-(tetrahydro-2H-pyran-4-yl)imidazo[1,2-b]pyridazin-6-amine.
  • This intermediate was prepared from 3-bromo-6-chloroimidazo[1,2-b]pyridazine and tetrahydro-2H-pyran-4-amine using Method D.
  • Step 2 3-Bromo-N-(cyclopropylmethyl)-N-(tetrahydro-2H-pyran-4- yl)imidazo[1,2-b]pyridazin-6-amine.
  • This intermediate was obtained by reacting the product obtained in Step 1 with (iodomethyl)cyclopropane using Method F. The crude product was directly used in next step after work up.
  • Step 1 3-Bromo-N-(4,4-difluorocyclohexyl)-N-methylimidazo[1,2-b]pyridazin-6- amine.
  • This intermediate was prepared from 3-bromo-6-chloroimidazo[1,2-b]pyridazine and 4,4-difluoro-N-methylcyclohexan-1-amine using the general Method D. Yield 38%.
  • Step 2 Reaction of the above intermediate with (1-methylindazol-6-yl)boronic acid using Method B afforded target compound 30. Yield 57%.
  • TLC R f 0.40 (CHCl 3 -MeOH, 9:1).
  • Step 2 Reaction of the above intermediate with N-methylpropan-1-amine using Method D afforded the desired target compound 33. Yield 28%.
  • TLC R f 0.40 (CHCl 3 - MeOH, 9:1).
  • Step 2 Reaction of the above intermediate with N-methylpropan-1-amine using Method E gave the target compound 49. Yield 33%.
  • TLC R f 0.40 (CHCl 3 -MeOH, 9:1).
  • This intermediate (200 mg, 0.6 mmol), bis(pinacolato)diboron (180 mg, 0.71 mmol), potassium acetate (116 mg, 1.18 mmol ), and 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) dichloride dichloromethane complex (50.5 mg, 0.06 mmol) in 1,4-dioxane (8 mL) was heated at 85 °C for 3 hours. The reaction mixture was poured into ethyl acetate (100 mL) and washed with water (3 x 50 mL) and saturated aqueous sodium chloride (1 x 50 mL).
  • Step 2 The above intermediate was reacted with 3-bromo-N-methyl-N- propylimidazo[1,2-b]pyridazin-6-amine using Method B. Yield 19%.
  • TLC R f 0.35 (CHCl 3 - MeOH, 9:1).
  • Step 2 The above intermediate (60 mg, 0.22 mmol) was dissolved in DMF (4 mL) and sodium hydride (10.7 mg, 60% dispersion in mineral oil, 0.27 mmol) was added. To the resulting solution was added tetrahydro-2H-pyran-4-ol (34 mg, 0.33 mmol) at 0 °C. The reaction mixture was stirred at room temperature overnight, diluted with EtOAc (20 mL), and washed with water (10 mL). The organic layer was dried and concentrated under reduced pressure. The residue obtained was purified via flash chromatography over silica using 0- 10% methanol in dichloromethane to obtain the desired compound. Yield 59%.
  • Step 1 6-Chloro-3-(1H-indazol-6-yl)imidazo[1,2-b]pyridazine.
  • This intermediate was prepared from 3-bromo-6-chloroimidazo[1,2-b]pyridazine and (1H-indazol-6-yl)boronic acid using Method B.
  • Step 2 The above intermediate was reacted with tetrahydro-2H-pyran-4-ol as described for the preparation of compound 57. Yield 58%.
  • HuR expression is associated with poor prognosis for glioma patients
  • the clinical outcomes of glioma patients harboring low or high expression of ELAVL1 (HuR) was analyzed by utilizing R2: Genomics Analysis and Visualization Platform (Jan Koster, Department of Oncogenomics, Academic Medical Center (AMC) Amsterdam, Netherlands).
  • ELAVL1 Low expression levels of ELAVL1 were associated with favorable prognosis (REMBRANDT Madhavan - 550 MAS.5.0-u133p2 study) as shown in FIG.1A; the expression levels of the ELAVL1 and ACTB (actin) as a control in the glioma samples (REMBRANDT Madhavan - 550 MAS.5.0-u133p2 study) compared to the normal brain samples (N Brain 44 Harris study) are illustrated in FIG.1B. There was significant ELAVL1 overexpression in the glioma group as compared to the normal brain group. Next, the influence of the ELAVL1 expression on the outcome of patients with different glioma grades was analyzed.
  • FIG.1D The ELAVL1 expression normalized to the ACTB expression in the tumors of different grades compared to the normal brain is shown in FIG.1D. There was an increase in average ELAVL1 expression with higher tumor grade. Next, a mini ontology analysis of gene sets was performed, which exhibited significant positive or negative correlations with the ELAVL1 expression, for normal brain and for each tumor grade based on R2: platform data, see FIG.1D.
  • Example 3 In vitro testing of inhibitors targeting HuR protein for oncogenic disease treatment. The oncogenic activity of HuR is driven by subcellular localization into the cytoplasm and formation of multimers of HuR. To develop cell based assays to characterize the ability of small molecules to disrupt this process, a split luciferase assay detecting the formation of HuR dimers was developed. The assays were performed in 96 well plates with clear bottom (Corning Inc., Corning, NY).
  • Triton X- 100 (0.3 %) in PBST buffer (i.e., PBS with 0.1% Tween 20) was used for cell permeabilization for 30 minutes at room temperature. Cells were rinsed with PBST buffer 3 times for 10 minutes after fixation. Blocking buffer (3% BSA, 22.52 mg/ml glycine in PBST) was used to block unspecific antibody binding for 30 minutes at room temperature. Primary cleaved caspase 3 antibody (Cell Signaling Technology; Danvers, MA) at 1:200 dilution in PBST buffer with 1% BSA was utilized overnight at 4°C for cleaved caspase 3 staining.
  • PBST buffer i.e., PBS with 0.1% Tween 20
  • cells were rinsed four times for 10 minutes with PBST buffer and then were incubated with secondary antibody (Alexa Fluor 594, goat anti-rabbit IgG, Invitrogen (Carlsbad, CA), 1:2000/5000 dilution) in PBST buffer with 1% BSA for 1 hour at room temperature in the dark.
  • secondary antibody Alexa Fluor 594, goat anti-rabbit IgG, Invitrogen (Carlsbad, CA), 1:2000/5000 dilution
  • PBST buffer 1% BSA for 1 hour at room temperature in the dark.
  • cells were rinsed with PBST buffer 4 times for 10 minutes in the dark.
  • Cell nucleus were stained with DAPI (4′,6-diamidino-2-phenylindole).
  • Cell images were obtained with EVOS Fl (Life Technologies; Eugene, OR) imaging system. The immunostaining on the brain tissue (fixed in paraformaldehyde, 4%) was performed.
  • tissue was permeabilized with 0.5% Triton X-100 in PBST buffer for 30 minutes, followed by three times wash with PBST and blocking with the Universal blocking buffer for 30 minutes. Then BEAT tm Blocker kit purchased from Zymed Laboratories (Carlsbad, CA) was utilized to block unspecific antibody binding to endogenous mouse IgG.
  • BEAT tm Blocker kit purchased from Zymed Laboratories (Carlsbad, CA) was utilized to block unspecific antibody binding to endogenous mouse IgG.
  • the primary antibodies anti-HuR from Santa Cruz Biotechnology (1:100; Santa Cruz, CA), anti-Bcl2 from Santa Cruz Biotechnology (1:50), and anti-Mcl1 (1:100) from Cell Signaling Technology were utilized overnight at 4oC in PBS (1% BSA) buffer.
  • the corresponding secondary antibodies Alexa Fluor 594 Goat anti-rabbit IgG (1:1000) from Invitrogen or Alexa Fluor 594 Donkey anti-mouse IgG (1:1500) from Life Technologies (Eugene, OR) were utilized in PBS (3% BSA) buffer for 1 hour at room temperature, followed by washing with PBST buffer four times.
  • DAPI was utilized for nuclear staining. The fluorescence signal was calibrated based on the secondary- only and primary-only control staining. Western blotting, antibodies, nuclear and cytoplasmic protein fractionations Western blotting was performed.
  • Nuclear and cytoplasmic protein fractionations were performed using NE-PER Nuclear and Cytoplasmic extraction reagents (ThermoFisher Scientific).
  • Anti-lamin A/C and anti-Tubulin alpha antibodies were utilized to verify nuclear and cytoplasmic fractions, respectively.
  • Antibodies against lamin A/C, cleaved PARP, cleaved caspase 3, SOX2, and Mcl1 were purchased from Cell Signaling Technology (Danvers, MA).
  • Anti-Tubulin alpha antibody was purchased from Sigma-Aldrich.
  • Antibodies against HuR, actin, and Bcl2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • mRNA isolation from adherent cell culture mRNA isolation and purification from adherent cells were performed using RNeasy Mini Kit and QIAshredder columns (Qiagen, Valencia, CA). Colony formation assays The soft agar colony formation assay was performed by using 0.9% and 0.45% agarose for the bottom and the top layers, respectively; cells were incorporated in the top layer. Seaplaque low-melting temperature agarose was purchased from Lonza (Rockland, ME). Five hundred cells per well were utilized in both, the soft agar and the attached colony formation assays, which were performed in 6 well plates; cells were treated with vehicle as the control or with the desired drugs, which were administered twice per week for three weeks.
  • the Crystal violet solution (0.1%) was utilized for colony staining in both assays. The colonies were counted after three weeks of treatment; the plate images were obtained by using Amersham Imager-600 reader (Piscataway, NJ).
  • Illumina global RNA-sequencing data Illumina global RNA-sequencing data were generated by utilizing PDGx neurospheres, which have been treated with a compound as described herein (e.g., SRI- 42127, 3 uM) for 12 hours or with vehicle as the control. RNA was isolated by using TRIzol reagent (Invitrogen; Carlsbad, CA). The RNA samples were processed.
  • Proteomic data The cell pellets from PDGx neurospheres, which have been treated with a compound as described herein (e.g., SRI-42127, 3 uM) for 18 hours or with vehicle as the control, were processed in the UAB CCC Mass Spectrometry/Proteomics shared facility.
  • the proteomic data were generated by using standard procedures.
  • Kinase profiling assays The kinase inhibitory potentials of compounds as described herein (e.g., SRI-42127 and SRI-41664) were evaluated by ThermoFisher Scientific's SelectScreen TM Kinase Profiling Service.
  • kinase inhibitory assays were utilized: ZLYTE (Madison, WI) for NTRK1, PIM1, and cMET kinases; LanthaScreen Binding (Madison, WI) for AAK1 kinase; and Adapta (Madison, WI) for IRAK1 kinase.
  • ZLYTE Modison, WI
  • LanthaScreen Binding (Madison, WI) for AAK1 kinase
  • Adapta Modison, WI
  • PK Pharmacokinetic assessment
  • SRI-42127 The in vivo PK study for SRI-42127 was performed by Pharmaron (Beijing, China); pharmacokinetics were evaluated in the C57Bl/6 mice.
  • Statistical analysis Statistical interpretations of cell cycle data, immunohistochemistry data, HuR/mRNA co-immunoprecipitation data, mRNA gene-specific quantitative data, western blotting data, colony formation data, cell viability data, results from in vivo experiments, transcript-specific data utilized in the enrichment analysis and the direct HuR mRNA target analysis were achieved by using a Student’s t test (when only two groups of data were analyzed) and one- way ANOVA with Turkey’s post hoc test (when multiple data groups were analyzed).
  • the assay used the coding sequence of HuR in frame with the N-terminus of firefly luciferase or the C-terminus as depicted in FIG.2A. When HuR forms dimers in this case, luciferase is generated and with luciferin present, luminescence is generated. A control is demonstrated in FIG.2B. The assay was used to determine the HuR IC 50 ( ⁇ M) of the compounds, as reported in the tables below, along with solubility at pH 7.4, Log D at pH 7.4, Mouse liver microsomal stability in vitro, and Human liver microsomal stability in vitro.
  • Table 1 Imidazo[1,2-b]pyridazines possessing various aryl substituents at the 3-position a Mouse liver microsomal stability in vitro. b Human liver microsomal stability in vitro. c A - symbol denotes that the compound was not tested. Table 2. Imidazo[1,2-b]pyridazines possessing 6-indazolyl and related substituents a 5, 19-36 37 38 aSee footnotes to Table 1. b Compound did not provide a clear solution in the assay medium. Table 3. Imidazo[1,2-b]pyridazines possessing 5-indazolyl and related substituents a 39-50, 53, 54 51, 52, 57 55, 56 aSee footnotes to Tables 1 and 2.
  • Compounds 39 and 40 are 5-indazolyl analogues of the 6-indazolyl compounds 5 and 19, respectively. These two 5-indazolyl analogues displayed IC 50 values of 0.3 ⁇ M and are 5– 8-fold more potent than the 6-indazolyl isomers. Among matched pairs, the 5-indazolyl isomers in general were more potent than the 6-indazolyl isomers. In the 6-indazolyl series, a methyl group at the 3-position (compounds 31 and 32) is not tolerated. Thus, in some cases, the compound is not an imidazo[1,2-b]pyridazine with a methyl group at the 3-postion.
  • the compound is not compound 31 or compound 32.
  • the placement of a methyl group at the 1-postion of the 5- indazolyl system is similarly not tolerated indicating a similar binding mode and steric encumbrance at the target site.
  • the compound is not compound 41 or compound 42.
  • compounds that displayed potent activity include 45, 48–50 possessing dimethylpyranylamino-, propylamino- , N-methylpropylamino- and trifluoroethylamino substituent at the 6-position of the imazopyridazine moiety.
  • a non-amino ether substituent such as the pyranyloxy substituent in both 5-indazolyl (57, Table 3 ) and 6-indazolyl systems (58) are tolerated yielding compounds with inhibition potencies of 2 ⁇ M and 3 ⁇ M, respectively.
  • Compounds that were identified as HuR inhibitors with IC 50 of less than 10 micromolar (uM) are listed in Table 4 and Table 5.
  • Table 4 HuR inhibitors with IC 50 ⁇ 10 uM
  • Squares represent luminescence signal from reporter representing HuR dimers (U251 cells co- expressing HuR-Nluc and HuR-Cluc constructs); circles represent luminescence signal from control reporter (U251 cells expressing Fluc construct). Results are shown as mean + SD.
  • a reduction of kinase inhibition potential was performed. This was achieved by the introduction of a methyl group on the pyrazolylamino and a pyranylamino NH resulting in compounds SRI-42124 and SRI-42127, respectively as shown in FIG. 3, Panel C.
  • Compound SRI-42124 had poor aqueous solubility that precluded compound evaluation in the cell-based assay.
  • the similar results were achieved in the four independent reporter cell clones overexpressing HuR-Cluc+HuR-Nluc constructs as shown in FIG. 4.
  • Compound SRI-42127 had reduced kinase inhibition potential compared to the parent SRI-41664 compound as shown in Table 4. Ultimately, compound SRI-42127 was established.
  • Example 4 Structural changes that led to compounds with HuR inhibition IC 50 values below 10 uM are presented in Table 4. In contrast, structural changes that led to compounds with HuR inhibition IC 50 values above 10 uM are presented in Table 5.
  • Example 4 ADME evaluations of compounds Selected compounds were assessed for their aqueous solubility, Log D and mouse and human liver microsomal stability. While favorable solubility and Log D could be achieved in several compounds, significant improvement in in vitro microsomal stability, particularly in mouse liver microsome, could not be achieved. From the group of compounds that showed potent HuR inhibition activity, a preliminary pharmacokinetic study in mice by ip administration was performed (in this case, for SRI-42127 (also referred to herein as “Compound 19”)).
  • FIG. 5A illustrates the inhibitory dose responses of SRI- 41664 and SRI-42127 compounds in the established U251, LN229, and the PDGx XD456 glioma cell lines.
  • An HuR-overexpression approach was employed to confirm SRI- 42127 inhibitory potency against HuR dimerization.
  • FIG. 7 Panels A-E illustrates HuR expression in established and PDGx glioma cell lines and the IC 50S of SRI-42127 compound to inhibit cell viability after 48 hours of treatment (data also include parental TMZ-resistant and stem cell lines).
  • HuR is one of the key regulators of cell cycle progression in cancer cells, particularly high-grade glioma; therefore, the arrest of cell cycle progression by the newly identified compounds supports HuR-targeted inhibition.
  • FIG.5B illustrates representative histograms of cell cycle progression in the established U251, LN229, and the PDGx XD456 glioma cell lines in the control (treatment with vehicles) and after treatment with SRI-42127.
  • the bar graph represents the average numbers of cells in each phase of the cell cycle after treatment with the SRI-42127 normalized to the corresponding control values (vehicle treatment). There was a significant accumulation of cells in the G1 phase and a reduction of cells in the S phase after treatment with SRI-42127, 10 uM for 18 hours.
  • FIG. 8A-D graphs illustrate the percent of reduction of Bcl2/18S and Mcl1/18S mRNAs following cell treatment with compounds as described herein versus control (vehicle treatment).
  • HuR protein plays a significant role in the stabilization of the mRNAs of the anti-apoptotic Bcl2-family, which contributes to the protection of genetically unstable glioma cells from apoptosis. Therefore, the Bcl2 family was evaluated in the established and PDGx cell lines after cell treatment with SRI-41664 or SRI-42127 compounds. A significant reduction in the expression of anti-apoptotic Bcl2 family molecules at the mRNA and protein levels after cell treatment with SRI-41664 or SRI-42127 compounds was observed.
  • the average decreases of Bcl2/18S and Mcl1/18S mRNA ratios were 96 ⁇ 2% and 94 ⁇ 3%, 96 ⁇ 2% and 78 ⁇ 4%, 88 ⁇ 2% and 75 ⁇ 4%, 93 ⁇ 1% and 84 ⁇ 10% based on three experiments in the established U251, U87, LN229, and the PDGx XD456 cell lines, respectively, after cell treatment with SRI-41664, 10 uM for 24 hours compared to the control.
  • the average decreases of Bcl2/18S and Mcl1/18S mRNA ratios were 96 ⁇ 2% and 95 ⁇ 3%, 95 ⁇ 2% and 86 ⁇ 7%, 95 ⁇ 2% and 87 ⁇ 8%, and 96 ⁇ 2% and 90 ⁇ 9% based on three experiments in the established U251, U87, LN229, and PDGx XD456 cell lines, respectively, after cell treatment with SRI-42127, 10 uM for 24 hours compared to the control.
  • FIG.8B representative western blots confirm reduction of Bcl2 and Mcl1 proteins in cytoplasmic fractions of U251 and XD456 cell lines following treatment with compounds as described herein, 10 uM for 48 hours.
  • SOX2 reduction in nuclear fractions indicates decrease of tumorigenicity of cell lines after treatment with compounds as described herein.
  • Actin and Lamin A/C were utilized for data normalization in cytoplasmic and nuclear fractions, respectively.
  • the representative western blots illustrate a dramatic reduction of Bcl2 and Mcl1 protein levels in the cytoplasmic fractions of both cell lines after treatment with SRI-41664 or SRI-42127 compounds compared to the control.
  • FIG.8C representative western blots illustrate appearances of cleaved PARP and cleaved caspase 3 in glioma cell lines after treatment with compounds as described herein, 10 uM for 48 hours.
  • a reduction in the expression of the anti-apoptotic Bcl2-family was accompanied by apoptosis: cleaved caspase 3 and cleaved PARP proteins.
  • the representative western blots illustrate the appearances of cleaved caspase 3 and cleaved PARP in the established U251 and PDGx XD456 glioma cell lines after treatment with SRI-41664 or SRI- 42127 for 48 hours.
  • FIG.8D Graphs in FIG.8D illustrate the average numbers of colonies formed by established U251 and PDGx XD456 cell lines in the attached cell colony formation assay and in the soft agar colony formation assay, which represents anchorage independent cell growth.
  • Example 6 Testing compound SRI-42127 in vivo
  • the half-life of compound SRI-42127 was short (0.16 hours) using a dose of 10 mg/kg by IP, see Table 7.
  • the impact of SRI-42127 on intracranial glioma growth in a mouse model was evaluated.
  • FIG. 9A PDGx XD456 cells expressing firefly luciferase and EGFP constructs were utilized for intracranial tumor formation.
  • mice were randomly divided into two groups after four and a half days of the intracranial tumor establishment and were treated with vehicle (control group) or SRI-42127 compound (SRI-42127 group), 15 mg/kg, twice a day for three weeks via intraperitoneal injection.
  • the new class of inhibitors of HuR dimerization (particularly compound SRI-42127) exhibited encouraging, however not yet statistically significant, glioma-inhibitory potential in vivo.
  • Putative binding model of SRI-42127 at HuR A computational docking study was performed to investigate the binding mode of the SRI-42127 compound at HuR. The apo structure of HuR was prepared from a crystal structure of RNA/HuR complex (PDB ID: 4ED5) using the Protein Preparation Wizard.
  • FIG. 9E illustrates the docked pose of SRI-42127 at HuR. According to the putative binding mode, SRI- 42127 can interact with four functionally important residues Y26, I103, R97, and R153 of HuR.
  • MM-GBSA an in silico binding free energy scoring approach, was used to estimate the binding affinity of SRI-42127 at HuR. Specifically, MM-GBSA scoring was performed on the fixed conformation of the docked pose of SRI-42127 at HuR using VSGB solvation model and OPLS3e force field. The same scoring was performed on the UMP (U8 from HuR co-crystal structure), which resides in the same region as SRI-42127 to obtain a reference value.
  • SRI- 42127 showed a stronger MM-GBSA binding free energy score than the UMP (-57.6 kcal/mol versus -22.7 kcal/mol), indicating that SRI-42127 is indeed capable of forming energetically favorable interactions at the RM1-RM2 putative binding site on HuR.
  • a mode of action includes the binding of SRI-42127 to RM1- RM2 induces a conformational change of HuR monomer that disrupts the optimal interface needed for HuR dimerization and mRNA binding.
  • Example 7 Effects of compound SRI-42127 on gene sets and cell signaling pathways RNA sequencing was performed on PDGx-derived glioma neurospheres representing the different molecular subtypes (classical, neuronal, and mesenchymal) after treatment with vehicle (control) or SRI-42127 (3 uM) for 12 hours. Analysis of direct HuR mRNA targets and their corresponding cell signaling pathways was performed. Table 9 illustrates the evaluated gene sets, representing direct HuR mRNA targets, and their corresponding cell signaling pathways. The gene set average values, which exhibited significant alterations following treatment with the SRI-42127 compound as compared to the control, are bolded.
  • FIG.10 demonstrates gene ontology enrichment analysis for gene sets significantly altered in PDGx neurospheres by SRI-42127, 3 uM 12 hours of treatment compared to a control (vehicle treatment).
  • Panel A illustrates enrichment of the significantly down-regulated cell signaling pathways/processes (chart on the left) and targeted subcellular structures (chart on the right) for significantly down-regulated gene sets after SRI-42127 treatment compared to control (vehicle treatment).
  • the RNA-Sequencing data was generated using glioma neurospheres representing the different molecular subtypes (classical, neuronal, and mesenchymal) in the control (treatment with vehicle) and after treatment with SRI-42127, 3 uM for 12 hours.
  • FIG.10 Panel A illustrates the enrichment for the significantly down-regulated cell signaling pathways and targeted subcellular structures.
  • the detailed cluster plots of the enriched down-regulated genes and corresponding cell signaling pathways and targeted subcellular structures are shown next to the corresponding charts.
  • FIG.10 Panel A illustrate the enriched down-regulated genes and corresponding cell signaling pathways and targeted sub-cellular structures detailed in Table 10 and Table 11. Table 10. Enriched down-regulated genes and corresponding cell signaling pathways Table 11. Targeted sub-cellular structures FIG.10, Panel B illustrates enrichment for significantly up-regulated cell signaling pathways and targeted subcellular structures.
  • ribosomal reorganization is the common adaptive response to translational and environmental stress, therefore the ribosome reorganizing pathways are considered the cell’s main signaling compensatory pathways, which were up- regulated in response to neurosphere treatment with the SRI-42127 compound.
  • the Illumina RNA-Sequencing data confirmed that SRI-42127 treatment induced up-regulation of Tubb2A transcript on more than 2 folds in four of five neurosphere cell lines, that supports low SRI-42127 cytotoxicity toward neurons and favors neuronal survival, see FIG.6.
  • the cluster charts of FIG.10, Panel B illustrate the enriched up-regulated genes and corresponding cell signaling pathways and targeted sub-cellular structures detailed in Table 12 and Table 13. Table 12. Enriched up-regulated genes and corresponding cell signaling pathways
  • the up-regulated protein sets exhibited several different patterns across cell lines at the evaluated time point; therefore, the significance of the up-regulated pathways on the protein level remains to be determined.
  • Example 8 In vitro and in vivo testing of inhibitors targeting HuR protein multimerization for inflammation disease treatment.
  • Neuroinflammation is a major driver of many central nervous system (CNS) diseases as well as secondary tissue injury related to trauma.
  • the two major cell types responsible for neuroinflammatory responses are microglia and astroglia. These cells become activated in response to CNS injury in a broad range of diseases and trauma, and this activation leads to disease progression or secondary tissue injury.
  • microglial and astroglial activation a major hallmark of microglial and astroglial activation is their production of secreted factors into the CNS milieu, including cytokines, chemokines, metalloproteases and other inflammatory mediators as shown in Table 14.
  • the secreted factors can produce toxic effects directly to neurons or indirectly by (a) increased vascular permeability of the blood-brain-barrier which leads to cerebral or spinal cord edema, (b) secondary ischemia due to small vessel occlusion or hemorrhage, (c) glial scar formation, (d) recruitment of other tissue-damaging immune cells through chemoattractive chemokines, (e) amplification of neuroinflammation through activation of recruited glial or peripheral immune cells which leads to further release of neurotoxic factors and substances.
  • Table 14 Secreted neuroinflammatory factors regulated by HuR in microglia and astroglia that drive disease progression
  • LPS lipopolysaccharide
  • Stimulation with LPS activates these cells to produce pro-inflammatory cytokines, mimics the neuroinflammatory profile associated with progression of neurological diseases and conditions described above, and is used for pharmacological screening.
  • HuR is predominantly a nuclear protein. An essential component to proper HuR function is its ability to translocate to the cytoplasm.
  • Cytoplasmic translocation of HuR in microglia and astroglia is a hallmark of activation. Blocking this translocation would impair HuR function and its ability to positively regulate disease- promoting secreted factors. Because HuR multimerization is a necessary process for cytoplasmic translocation, inhibiting its multimerization blocks translocation.
  • Experimental details Cell LPS activation and analysis Cultured primary microglial cells isolated from neonatal mouse brain were treated with LPS to achieve activation. Cells were then treated with varying doses of SRI-42127 or vehicle. Imagestream single cell flow cytometry was used to track the location of HuR within microglial cells as depicted in FIG.11, Panel A. DAPI was used as a flow marker for nuclear localization.
  • the percentage of HuR immunofluorescence that colocalized with the nuclear compartment versus the cytoplasmic compartment was quantified.
  • DMSO unstimulated state
  • the characteristic nuclear predominance of HuR was observed as reflected by a low percentage of cells with high HuR in the cytoplasm.
  • LPS stimulation there was an ⁇ 8-fold increase in the percent of cells with high levels of HuR in the cytoplasm ( ⁇ 60%).
  • SRI-42127 treatment there was a significant dose-dependent decrease in cells with high cytoplasmic HuR. At 1 ⁇ M of SRI-42127, the percent of cells with high HuR was similar to resting (unactivated) microglia ( ⁇ 10%).
  • HMGB1 is nuclear predominant and translocates with microglial activation. Primary microglia were treated with LPS and 0.5 ⁇ M of SRI-42127 for 24 hours. Cells were fixed and immunostained for HMGB1 and IBA1. Nuclear/cytoplasmic localization was assessed by confocal microscopy.
  • HMGB1 immunofluorescence in each cell was quantified using Fiji software. The nuclear portion was determined by quantifying the amount of HMGB1 fluorescence that overlapped with DAPI fluorescence. HuR translocation was studied in parallel with the same methodology. In unactivated microglia (LPS-), both HuR and HMGB1 were nuclear predominant with little to no merged signal with cytoplasmic IBA1 as depicted in FIG.11, Panel C. The calculated nuclear/cytoplasmic (N/C) ratio was between 1.5 and 2 as shown in FIG.11, Panel C and FIG.11, Panel D.
  • FIG.18 In vehicle-treated animals, there was translocation of HuR into the cytoplasm as indicated by a merged yellow signal whereas in SRI-42127 treated mice, there was little to no merged signal. Representative photomicrographs are shown in FIG.18 (top). Next, 20-30 IBA1+ cells from sections in 3 control and 3 treated mice were assessed. A 2-fold increase in the N/C ratio of mice treated with SRI-42127 was observed, confirming the effect of the drug as shown in FIG.18 (bottom). Next, the activation state of microglia was studied by measuring IBA1 intensity. Sections from the hippocampal region were stained with IBA1 and fluorescence intensity was measured using Fiji software from 5 sections of 3 mice per group. The reviewer was blinded to the identity of the samples.
  • FIG.20 Panel B.
  • Each region plus a sample from the whole brain were studied.
  • SRI-treated mice a significant attenuation of infiltrating neutrophils (2 to 3-fold) and monocytes (1.3-to 2 fold) in all brain regions was observed as shown in FIG.20, Panel C.
  • HuR KO mice there was significant attenuation of monocytes but not neutrophils as showin in FIG.20, Panel D.
  • SRI-42127 produces neuroprotective effects in a study of spinal cord injury and attenuates chronic pain in a peripheral nerve injury model. Wild-type male mice were subjected to a mid-thoracic contusion injury and then treated with SRI-42127 (15 mg/kg) every 8 hours for 4 days, starting 1 hour after injury. Assessment of recovery using the Basso motor scale (BMS) and horizontal ladder test (HLT), showed improvement in function with SRI-42127 as shown in FIG.21, Panel A.
  • BMS Basso motor scale
  • HLT horizontal ladder test
  • mice underwent sciatic nerve lesioning to produce a spared nerve injury and then received SRI- 42127 (15 mg/kg) or vehicle every 6 hours for 4 days (starting 1 h after injury). Mechanical sensitivity was assessed at day 7 post-injury.
  • the control (DMSO-treated) group showed typical allodynic responses, whereas the test group (SRI-42127-treated) showed preserved mechanical sensitivity (p ⁇ 0.001, FIG.21, Panel C).
  • SRI-42127 or DMSO

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Abstract

La présente divulgation concerne des composés qui inhibent des protéines de liaison à l'ARN, telles que la protéine R d'antigène humain (HuR). Les composés selon l'invention ont une affinité élevée pour les multimères HuR et inhibent les processus pathologiques qui favorisent le cancer et l'inflammation. Les composés sont hautement solubles dans l'eau et ont une bonne biodistribution pour les processus pathologiques systémiques et du système nerveux central. Les composés fournissent une option thérapeutique unique pour des processus pathologiques liés à la progression néoplasique ou à une inflammation aiguë ou chronique.
PCT/US2021/028460 2020-04-21 2021-04-21 Inhibiteurs de la multimérisation de protéine de liaison à l'arn et leurs procédés d'utilisation WO2021216757A1 (fr)

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WO2023220967A1 (fr) * 2022-05-18 2023-11-23 Anheart Therapeutics (Hangzhou) Co., Ltd. Procédé de production de composés d'imidazo[1,2-b]pyridazine 3,6-disubstitués

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